Lipid aggregates such as liposomes can function to facilitate introduction of macromolecules, such as DNA, RNA, and proteins, into living cells. Recently, it has been shown that lipid aggregates comprising cationic lipid components can be especially effective for delivery and introduction of large anionic molecules, such as nucleic acids, into certain types of cells. See Felgner, P. L. and Ringold, G. M. (1989) Nature 337:387-388. Since the membranes of most cells have a net negative charge, anionic molecules, particularly those of high molecular weight, are not readily taken up by cells. Cationic lipids aggregate to and bind polyanions, such as nucleic acids, tending to neutralize the negative charge. The effectiveness of cationic lipids in transfection of nucleic acids into cells is thought to result from an enhanced affinity of cationic lipid-nucleic acid aggregates for cells.
A variety of types of lipid aggregates are known, including liposomes, unilamellar vesicles, multilamellar vesicles, micelles and the like, having particle sizes in the nanometer to micrometer range. As is well-known in the art, the structures of lipid aggregates depend on the lipid composition and the method employed to form the aggregate. Cationic lipids can be used alone or in combination with non-cationic lipids, for example with neutral phospholipids like phosphotidylethanolamines, to form positively charged vesicles and other lipid aggregates which are able to bind nucleic acids. The positively charged lipid aggregates bind to nucleic acids, can then be taken up by target cells and thus facilitate transfection of the target cells with the nucleic acid. (See, Felgner, P. L. et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7417: Epstein, D. et al. U.S. Pat. No. 4,897,355.)
Cationic lipids are not universally effective for transfection of all cell types. Effectiveness of transfection of different cells depends on the cationic lipid composition used and the type of lipid aggregate formed. In addition, a particular cationic lipid may be more or less toxic to a given cell line, limiting the type or concentration of lipid that can be employed for transfection. Certain types of higher eukaryotic cells are not readily transfected employing presently available cationic lipids. These hard to transfect cells generally include suspension cell lines and primary human cell lines and more specifically include fibroblasts and macrophage cell lines. Compositions and methods which would generally enhance the efficiency of cationic lipid-mediated transfection and/or broaden the range of cell types that can be efficiently transfected with cationic lipid-DNA complexes would represent valuable improvements in the art.
Many biological materials are taken up by cells by receptor-mediated endocytosis. See: Pastan and Willingham (1981) Science 214:504-509. This mechanism involves binding of a ligand to a cell-surface receptor, clustering of ligand-bound receptors, and formation of coated pits followed by internalization of the ligands into endosomes. Both enveloped viruses, like influenza virus and alphaviruses, and non-enveloped viruses, like adenovirus, infect cells via endocytotic mechanisms. See: Pastan, I. et al. (1986) in Virus Attachment and Entry into Cells, (Crowell, R. L. and Lonberg-Holm, K., eds.) Am. Soc. Microbiology, Washington, p. 141-146; Kielian, M. and Helenius, A. (1986) "Entry of Alphaviruses" in The Togaviridae and Flaviviridae, (Schlesinger, S. and Schlesinger, M. J., eds.) Plenum Press, New York p. 91-119; FitzGerald, D. J. P. et al. (1983) Cell 32:607-617. Receptor-mediated endocytosis has been exploited to deliver DNA into cells. Wu, G. Y. and Wu, C. H. (1987) J. Biol. Chem. 262:4429-4432; Wagner, E. et al. (1990) Proc. Natl. Acad. Sci. USA 87:3410-3414. These methods employ bifunctional conjugates having a ligand, which binds to a specific cell-surface receptor, covalently linked to a DNA-binding domain. Asialoglycoprotein-polylysine conjugates and human transferrin-polylysine conjugates have, for example, been demonstrated to mediate DNA entry into certain eukaryotic cells. (Wagner, E. et al., 1990, supra).
Curiel, D. T. et al. (1991) Proc. Natl. Acad. Sci. USA 88:8850-8854 and Cotton, M. et al. (1992) Proc. Natl. Acad. Sci. USA 89:6094-6098 have recently reported that receptor-mediated transfection via transferrin-polylysine/DNA complexes is enhanced by simultaneously exposing the cells to defective adenovirus particles. These authors report that adenovirus particles function to disrupt endosomes containing the viral particle and the DNA complex. Replication-defective adenovirus particles and psoralen inactivated adenovirus were reported to enhance transfection. Adenovirus enhancement of transfection is limited, however, to cells which have both a ligand receptor, i.e. transferrin receptor, and an adenovirus receptor. Direct coupling of polylysine/DNA complexes to adenoviruses has also been employed for transfection. Curiel, D. T. et al. (1992) Hum. Gene Therapy 3:147-154; Wagner, E. et al. (1992) Proc. Natl. Acad. Sci. USA 89:6099-6103. In related work, Wagner, E. et al. (1992) Proc. Natl. Acad. Sci. USA 89:7934-7938, report augmentation of transfection in several cell lines when hemagglutinin HA-2 N-terminal fusogenic peptides from influenza virus are included in transferrin-polylysine-DNA complexes. The use of influenza peptide conjugates was, however, reported to be less effective for enhancement of transfection than defective adenovirus.
PCT patent applications WO 93/07283 and WO 93/07282, both published Apr. 15, 1993, relate to transfection of higher eukaryotic cells via ligand/polylysine/DNA complexes and endosomolytic agents, such as adenovirus and HA-2 fusogenic peptides.
Alphaviruses, mosquito-transmitted members of the family Togaviridae, are RNA-containing enveloped viruses (also called membrane viruses). Alphaviruses include, among others, Sindbis and Semliki Forest (SFV) viruses, several equine encephalitis viruses (Eastern (EEE), Western (WEE) and Venezuelan (VEE)), Chikungunya virus and Ross River virus. Sindbis and Semliki Forest viruses are the least virulent and best characterized alphaviruses. See generally: Schlesinger, S. and Schlesinger, M.J., eds. (1986) The Togaviridae and Flaviviridae, Plenum Press. Alphaviruses in general, and specifically SFV and Sindbis virus, have very broad host ranges. SFV infects a wide variety of cultured cells including mammalian (human, monkey, hamster, mouse, porcine), avian, reptilian, amphibian and insect cell lines. Liljestrom P. and Garoff, H. (1991) Biotechnology 9:1356-1361 and references cited therein. Animal cell expression vectors have been based on SFV (Liljestrom and Garoff (1991), supra) and Sindbis virus (Xiong, C. et al. (1989) Science 243:1188-1191). The entry of alphaviruses into cells has been studied using SFV as a model. Kielian and Helenius (1986) supra. As with other viruses, SFV binds to the cell membrane, and is internalized in coated vesicles. In contrast to non-enveloped viruses, SFV (and other enveloped viruses) is released into the cell cytoplasm by fusion of the viral envelope with the endosome membrane. Acidic pH triggers the fusion process. The fusion process in SFV is characterized as rapid, non-leaky and strictly dependent, both in in vitro fusion with liposomes and in vivo infection, on the presence of a 3.beta.-OH sterol, such as cholesterol, in the membrane to which the virus fuses. Kielian, M. and Helenius, A. (1985) J. Cell Biol. 101:2284-2291; Kielian, M. and Helenius, A. (1984) J. Virol. 52:281-283; White, J. and Helenius, A. (1980) Proc. Natl. Acad. Sci. USA 77:3273-3277; Phalen, T. and Kielian, M. (1991) J. Cell. Biol. 112:615-623. Although the detailed mechanism of fusion in alphaviruses (SFV and Sindbis virus) is not completely understood, alphavirus fusion is reported to be distinct from that of influenza virus. Kielian and Helenius (1985) supra; Wahlberg J. M. et al. (1992) J. Virol. 66:7309-7318. As noted above, fusion of the influenza virus is associated with influenza hemagglutinin (HA). An acidic pH-induced conformational change in HA exposes a hydrophobic domain, containing N-terminal sequences of the HA-2 subunits, which is thought to bind to the target membrane facilitating fusion. SFV spike glycoprotein is distinct in size, structure and amino acid sequence from HA and does not have a hydrophobic domain linked to fusion as does HA.
In addition to the togaviruses (e.g., alphaviruses) and orthomyxoviruses (influenza), enveloped viruses include the following major families of animal viruses: Herpesviridae, Bunyaviridae, Paramyxoviridae, Rhabdoviridae, Retroviridae, Arenaviridae, Coronaviridae and some members of Iridoviridae. Although all enveloped viruses are released into the cell cytoplasm by fusion of the viral envelope with the outer cell membrane, the specific fusagenic component and thus mechanism of fusion may vary. For example, vesicular stomatitis virus (VSV), a rhabdovirus, infects host cells via adsorptive endocytosis. See, e.g., Dahlberg, J. E. (1974) Virology 58:250-262; Dickson, R. B. et al. (1981) J. Cell Biol. 89:29-34; Fan, D. and Sefton, B. (1978) Cell 15:985-992; and Matlin, K. S. et al. (1982) J. Mol. Biol. 156:609-631. VSV fusion is thought to involve interaction between the VSV glycoprotein (G protein) and specific membrane lipids (Schlegel, R. et al. (1983) Cell 32:639-646). The VSV G protein reportedly binds preferentially to "saturable receptors" such as acidic phospholipid phosphatidylserine (Schlegel, R. and Wade, M. (1985) J. Virol. 53(1):319-323. Unlike the fusion process for SFV, VSV fusion does not require the presence of a 3.beta.-OH sterol. See: Young, J. D. E. et al. (1983) Virology 128:186-194, and Phalen, T. and Kielian, M. (1991) supra.
The present invention is based on the discovery that components of enveloped viruses can significantly enhance the efficiency of cationic lipid-mediated transfection of eukaryotic cells. Unlike prior art methods, the enhanced transfection methods of this invention do not require encapsulation of the nucleic acid within anionic phospholipid-based liposomes. The present invention thus eliminates the need to construct liposomes for each particular nucleic acid, an inconvenient and often difficult procedure. Moreover, the enhanced transfection methods of this invention do not require conjugation of a polycation to a ligand, nor to the virus itself. The methods of this invention are applicable to a wider range of cell-types than prior art methods. There is no requirement for specific ligand receptors in target cell lines. Furthermore, since the alphaviruses, at least SFV, require only cholesterol or closely related sterols in target cells, the range of cells to which the methods of this invention can be applied is much broader than prior art methods. In addition, the methods of this invention can be combined with techniques well-known in the art for introducing cholesterol into cell membranes or enhancing the level of cholesterol in cell membranes to further enhance transfection efficiency or further broaden the range of cell types to which these methods are applicable.